Higher-order chromatin organization is an essential aspect for the expression of eukaryotic genes. The gypsy chromatin insulator may establish chromatin domains that are thought to regulate gene expression by controlling the organization of DNA within the nucleus. However, the mechanism governing the establishment and maintenance of this chromatin organization is not well understood. I have identified Topoisomerase II as a potential regulator of insulator complexes and higher-order chromatin structure. This proposal seeks to better define this role for Topoll as it relates to insulator function. Novel Topoll mutants will be characterized and genetically tested for their interaction with insulator function. In addition, the Topoll protein will be examined for its association with insulator complexes in vivo. Finally, a biochemical approach will be undertaken to better define Topoll interaction with insulator DNA and proteins. From these studies I hope to better define the connection of Topoll as it pertains to insulator function and higher-order chromatin organization. [unreadable] [unreadable]